Screening method for predicting susceptibility to breast cancer

ABSTRACT

A method to aid in identifying a familial or sporadic pattern of risk in at least one individual for developing cancer of a mucosal epithelial tissue, the method comprising screening said at least one individual for heterozygosity or homozygosity for a mutation in a gene coding for a Poly-Ig (Fc) receptor or a Poly-Ig-like (Fc) receptor capable of mediating inhibition of cancer cell growth by an immunoglobulin inhibitor. A method of treating an individual so identified includes enhancing the amount of immunoglobulin inhibitor contacting a mucosal epithelial tissue of said individual, and, especially in individuals homozygous for the defective receptor, may also include prophylactic surgery. Other methods include implementation of a risk reduction or prevention program in individuals identified as being at risk.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application No. 60/332,920 filed Nov. 14, 2001, andis a continuation-in-part of U.S. patent application Ser. Nos.09/852,958 and 09/852,547, both filed May 10, 2001, the disclosures ofeach of which are hereby incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Research leading to the present invention was supported in part by thefederal government under Grant Nos. DAMD17-94-J-4473, DAMD17-98-1-8337and DAMD17-99-1-9405 awarded by the Defense Department through the USArmy Medical Research and Materiel Command, Breast Cancer ResearchProgram. The United States government may have certain rights in theinvention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to genetic changes associatedwith the onset of sporadic breast cancer, and more particularly tomethods and compositions for identifying such changes and for screeningwomen at risk of developing the disease.

2. Description of Related Art

The number of breast cancer cases diagnosed each year worldwide is aboutone million (1). Breast cancer represents 18% of all cancers in womenand, with the exception of skin cancer, is the most common site ofcancer in women. In decreasing order, the incidence of other cancers inwomen are cervix, colon/rectum, stomach, endometrium, lung, ovary,mouth/pharynx, esophagus and lymphoma (1). The fact that a similarpattern is widespread throughout the Western world suggests that relatedgenetic changes may be the origin of cancer development at these sites.

Many risk factors have been defined for breast cancer (1-10). Whenspecific risks are given relative ranks, an interesting pattern emerges.The major four risk factors are age (relative risk 10) followed bygeographic location, previous breast cancer, and previous benign breastdisease (relative risks of 4 to 5) (1). Reproductive history is nextmost important with relative risks of 2 to 3 (1). Other factors such asdiet, alcohol consumption, socioeconomic group and family history are inthe relative risk range of 1.3 to 2 (1). Notably, the risk of oralcontraceptives or hormone replacement therapy is low, in the relativerange of 1.2 to 1.4 (1). From these data, it can be concluded that theorigin of breast cancer, and hence the number of causative genes, areyet to be identified. While modification of personal habits,reproductive considerations, and behavior can reduce risk, the benefitsare modest and certainly offer no guarantees. Thus far, traditionalepidemiology has not provided “the” origin of breast cancer. Indeed,based on epidemiological data, it has even been suggested that breastcancer arises from a single cause (11). But this conclusion came withthe statement that the cause was still unknown. With the growingimportance of genetic analysis, it now appears likely that epidemiologywill move into this arena to make further advances.

Other investigators have made observations that are critical tounderstanding the genetic origins of breast cancer. First, it is clearthat only a small minority of cancers originate from germ-line mutations(12). Second, there are more than 100 changes in gene expressiondetectable in breast cancer cells versus normal breast epithelium (12).This large number promises to increase to 200 to 300 with new technology(13,14). Based on a recent discussion (12), cancer mutations have beendefined as Class I, which involve changes in gene DNA sequences, andClass II genetic alterations, in which changes in gene expression aredetected by mRNA analysis (12). By selecting specific RNA species forfurther study, information can be gained, but there is no assurancewhich change(s) is causative. Indeed, it seems highly unlikely thatcancer development requires this huge number of gene alterations. It ismore likely that most of the changes are the result of malignanttransformation, not the cause. It may be possible to use mass geneexpression analysis (e.g. microarray technology) to predict breastcancer risk or susceptibility, but for now this seems distant.

It seems reasonable to retreat from these types of “shotgun” analysisand approach the issue from another perspective. In this proposal, focusis placed on genetic changes that are more subtle and are represented byloss of heterozygosity (LOH) or other allelic imbalances (AI). Thesegenetic alterations are known to be associated with a high risk ofcancer development. There are categories of women with three or moretimes higher risk of developing breast cancer than average. They areoften classified into a group termed “familial breast cancer”. They have(i) a first degree relative with breast or ovarian cancer, (ii) onefirst degree relative with disease diagnosed under the age of 40, (iii)two first or second degree relatives with breast or ovarian cancer underthe age of 60, or (iv) three first or second degree relatives withbreast or ovarian cancer on the same side of the family (1). It istempting to conclude that this represents an inherited trait or at leasta propensity to development of the disease. This conclusion must betempered however by an understanding that “inherited” might include asyet unrecognized non-biological inheritance such as culture inheritanceand common environmental conditions (15). While genetic predispositionremains a strong possibility, it may not be of the type seem with BRCA1(16) and BRCA2 (17) which are inherited as autosomal dominants fromeither parent, albeit with varying penetrance. Equally, it is possiblethat the predisposition is a recessive inheritance, also with varyingpenetrance, or a recessive mutation that leads to breast cancerdevelopment. It may also represent inherited or acquired geneticabnormalities such as loss of heterozygosity (LOH) or other allelicimbalances (AI) such as abnormal gene numbers.

A familial aspect to breast cancer has been recognized for some time.However, this term may have different meanings. It might indicate“familial clustering” which is the existence of several cases in anextended family. Because breast cancer will occur in 10% of women,chance alone will allow some familial clusters depending upon the numberof females in a related group. Therefore, because a family has more thanone member with the disease, it does not necessarily follow that thereis a genetic cause. In contrast, the familial aspect of breast cancermight also indicate “familial aggregation” which is increased risk toclose relatives of women with breast cancer compared to relatives ofwomen without the disease. Familial aggregation is another matter. Itsexistence may depend on genetic and/or non-genetic causes. As discussedabove, the presence of breast cancer in one or more first degreerelatives increases risk significantly. As the number of breast cancersin first and second degree relatives increases, especially at youngerages, there is strong reason to hold that there are familial factorsthat may be genetic. If genetic in origin, identification of these geneswill be a major advance in understanding breast cancer (18). Genes whichshow LOH or AI are strong candidates for identification. The compositionand methods of identification of such genes is a primary focus of thisdisclosure.

The question is: “What genes are being sought to explain familialaggregation”? The low frequency of BRCA1 and BRCA2 does not support theview that they account for one million new cases each year worldwide(18). Although somewhat difficult to assess, one report indicates anestimated frequency of significant mutations in these two genes in theUS and UK of 0.0005 to 0.002 (19). Another report (25) states mutationsin 1 in 152 and 1 in 833 for mutations in these two genes. It is higherin some regions of the world such as Scandinavia and approaches 1 in 40in Ashkenazi Jews (20). The major issues resulting from the lack ofbroad application of BRCA1 and BRCA2 are what other genes are to besought and by what methods.

There are three separate genetic syndromes that are associated withabove average rates of breast cancer development (26). These areLi-Fraumeni Syndrome, Cowden's Syndrome and Ataxia Telangiectasia (AT).Each appears to involve a different gene lesion or small set of lesions,but nonetheless all contribute to a higher risk of breast cancer. Thesesyndromes illustrate the point that a small number of critical mutationsare likely involved in breast cancer development. In addition, LynchSyndrome II is associated with breast cancer development (28). Thegenetic lesion is important in Lynch Syndrome because it involvesimpairment of the critical process of DNA mismatch repair (29,30).

Li-Fraumeni Syndrome (LFS) is a very rare germ-line mutation in p53 (31)that increases premenopausal breast cancer as well as sarcomas, braintumors, leukemia and adrenocortical cancer (32, 33). LFS is an autosomaldominant syndrome for which genetic testing is not yet available. It isthought that availability would not change medical management (34).Nonetheless, p53 mutations occur in 30% of sporadic breast cancers (27).The very diverse types of p53 mutations in breast cancer pose a problemfor genetic testing (27). One study suggests that mutations at codon 248might be associated with higher breast cancer risks (35). Whilescreening for P53 changes alone may not be productive, combinationscreening of P53 with other genes may be very informative based on theaccepted view that cancer development is a multistep process involving arelatively few genes.

Cowden's Syndrome is characterized by excess breast cancer,gastrointestinal malignancies, thyroid disease, and other benignconditions (36). Cowden's syndrome carries a lifetime breast cancer riskof 25 to 50%. There is usually early onset and often appearance ofbilateral disease (37). There is a germ-line mutation in PTEN, a proteintyrosine phosphatase with homology to tensin. PTEN acts as a tumorsuppressor and functions in the control of the cell cycle (38). As withP53, screening for PTEN mutations alone may not be informative, but incombination with other genes, may provide important prognostic value.

Ataxia Telangiectasia (AT) is an autosomal recessive disordercharacterized by neurologic deterioration, telangiectasias,immunodeficiencies, and marked hypersensitivity to ionizing radiation.Approximately 1% of the population may be heterozygote carriers of theAT mutation (ATM). Over 200 mutations have been identified in the ATM.The majority are truncations (39). ATM proteins have a role in cellcycle control (40). ATM individuals are susceptible to cancer. Studieshave shown that even heterozygotes are at elevated risk for breastcancer (41, 42). This is the case despite the fact that the ATM is notidentified in breast cancer specimens in excess of its occurrence incontrol populations (43-45). The link between ATM and breast cancer maybe related to the kinase coded for by the AT gene. Its absence leads tochromosomal instability, a condition often associated with breastcancer. Because of the relative high frequency of heterozygotes in theU.S. and European populations, analysis of the AT gene plus other geneswill be useful.

Lynch Syndrome II is a form of several related diseases first reportedas “cancer family syndrome” (46). Other types of this disease are LynchSyndrome I, (also called hereditary nonpolyposis colon cancer—HNPCC),Miur's Syndrome (also called Torre's Syndrome), Down family Syndrome,Bloom's Syndrome and finally Dyskeratosis congenital (46). For thepurposes of example, Lynch Syndrome II will be discussed. Thisdiscussion is intended to encompass these other related forms of thedisease as they relate to breast cancer development. Lynch Syndrome IIis accompanied by the aggregation of colon, endometrial, ovarian andbreast cancer in families (28). This disease is due to mutations in DNAmismatch repair genes designated hMSH2 and hMLH1 (47). The result ofthese mutations is to create microsatellite instability (48).Microsatellite instability is used today to measure the mutations inpopulations of specimens from colon cancer patients (49). This has givena range of estimates of the size of population bearing these mutations.It seems likely that the two mutations lead to the accumulation ofmutations throughout the genome. With time, genes important in growthregulation of mucosal cells become altered and result in the onset ofcancer.

Cancer is now generally thought to be a multistep disease that arises inresponse to genetic changes altering key regulatory proteins within thecells. The mutations leading to cancer can be present in the germ lineor can arise as somatic mutations in the tissues. It is clear that aprogression exists whereby normal cells change to arrive at the fullymalignant state capable of metastasizing to distant body sites. Whilemany gene expression changes can be detected by sophisticatedtechnology, it is reasonable to conclude that the powerful methodsapplied mask the fact that only a relative few changes ultimately resultin cancer.

Although advancement has been made toward understanding geneticpredisposition to development of certain breast cancers, there remains apressing need for ways to identify the genetic changes associated withthe onset of sporadic breast cancers, which represent about 60-70% ofthe total number of cases diagnosed each year that have no known geneticorigin. There is also a great need for ways to screen individuals forrisk of developing the disease and for taking appropriate preventativemeasures.

SUMMARY OF PREFERRED EMBODIMENTS

The present invention addresses the problem of sporadic breast cancer,which represents about 60 to 70% of the total cases diagnosed each yearand has no known genetic origin. New methods and compositions areprovided which employ a newly identified Poly-Ig (Fc) receptor or aPoly-Ig-like (Fc) receptor that is characterized by, among other things,its ability to mediate the recently discovered cell growth inhibitoryfunction of the secretory immune system (e.g., dimeric/polymeric IgA andpolymeric IgM). These discoveries are described in detail in U.S. patentapplication Ser. Nos. 09/852,547 and 09/852,958, and in InternationalPatent Application Nos. PCT/US01/15171 and PCT/US01/15183, thedisclosures of which are hereby incorporated herein by reference.

In accordance with certain embodiments, methods and compositions forcarrying out genetic analysis of the Poly-Ig (Fc) receptor or aPoly-Ig-like (Fc) receptor to determine breast cancer susceptibility inheterozygotes and homozygotes are provided. In some embodiments of thepresent invention, gene screening compositions and methods are providedfor identifying women at risk of developing the disease either through afamilial pattern or a sporadic pattern. In some embodiments, methods andcompositions for double screening an individual for Poly-Ig (Fc)receptor or Poly-Ig-like (Fc) receptor mutations and for mutations ofother breast cancer predisposing genes are provided. In certainembodiments of the invention, methods and compositions for screening anindividual for heterozygous ATM mutations are provided which includecomparing the results to screening results for mutations in the Poly-Ig(Fc) receptor or Poly-Ig-like (Fc) receptor.

In some embodiments, methods and compositions for screening breastcancer specimens for mutations in growth regulating genes are provided.In another embodiment of the present invention, methods and compositionsfor evaluating breast fluid derived cells for molecular changes inpredetermined genes. In some embodiments, premalignant changes in thePoly-Ig (Fc) receptor or a Poly-Ig-like (Fc) receptor gene are assessed,and changes in other predetermined breast cancer predisposing genes arealso evaluated.

Also provided in accordance with the present invention is an analyticmethod that permits a determination of breast cancer susceptibility orrisk of future disease. The method includes genetic testing for loss ofheterozygosity (LOH) and other Allelic Imbalances (AI) in the D1S58locus of chromosome 1. In some embodiments, the analysis is done withblood cells or mucosal scraping cells to test for germline alterations.For example, analyses are carried out with samples from young women orthose at risk because of first or second degree relative breast cancerpatterns. Performing early genetic analysis is highly desirable foridentifying women at risk. If germline mutations are found or if somaticmutations are found, an appropriate “risk reduction” or “prevention”technology as described in U.S. patent application Ser. No. 09/852,547and in International Patent Application No. PCT/US01/15171 (alsoidentified as item 21 in the References, below), or in co-pending U.S.patent application Ser. No. ______ (Atty. Dkt. No. 1944-01201), entitled“Breast Cancer Eradication Program,” can be timely applied. Additionalpreventative or risk reduction methods and compositions are described inco-pending U.S. patent application Ser. No. ______ (Atty. Dkt. No.1944-01301), entitled “Anti-estrogen and Immune Modulator Combinationsfor Treating Breast Cancer.”

In other embodiments, similar analyses are done with breast fluidderived cells to identify somatic mutations. In this way somatic changesare revealed that may be classified as “pre-malignant” and hence usefulin identifying risk in the relative near future. In other embodiments,genetic analysis of breast tumors are carried out as an aid todetermining the type of cancer present and indicating if immune therapyis appropriate.

In still other embodiments, a method of treating an individualidentified as heterozygous or homozygous for a mutation in a gene codingfor a Poly-Ig (Fc) receptor or a Poly-Ig-like (Fc) receptor capable ofmediating estrogen reversible inhibition of breast cancer cell growth byan immunoglobulin inhibitor is provided. After an at-risk individual isidentified, using the an above-described screening method, treatmentincludes enhancing the amount of immunoglobulin inhibitor (“immunemodulator”), or an immune modulator mimicking compound, contacting amucosal epithelial tissue of said individual. This can be done by eitherboosting the body's natural secretory immune system (e.g. by way of animmune enhancing agent such as, Levimisol, Imiquimod, Picibanil andDHEA) or by administering a pharmaceutical composition containing activeforms of the immunoglobulin inhibitors or a drug that mimics itsinhibitory activity. In some embodiments, an anti-estrogen compound,such as tamoxifen, for example, is also administered to the individual.In some embodiments a hormone is co-administered with an anti-estrogenor immune modulator to reduce side effects of the drug. In someembodiments an aromatase inhibitor is also administered to theindividual. In some embodiments the treatment also includes prophylacticsurgical removal of non-cancerous breast tissue.

These and other embodiments, features and advantages of the presentinvention will become apparent with reference to the followingdescription.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Recent discoveries disclosed in co-owned, co-pending U.S. patentapplication Ser. Nos. 09/852,547 and 09/852,958, and in InternationalPatent Application Nos. PCT/US01/15171 and PCT/US01/15183 (alsoidentified as items 21 and 22 in the References, below), each of whichis hereby incorporated herein by reference, are expected to lead toresolution of the problem of how to identify genes that are key to the60 to 70% of breast cancer cases that are today termed “sporadic.” Theisolation of new “serum factor(s)” that regulate estrogen responsivebreast cancer cell growth in culture is described in the precedingapplications. The purification yielded dimeric/polymeric immunoglobulinA (IgA) and pentameric immunoglobulin M (IgM) as the active regulators.These immunoglobulins (“immunoglobulin inhibitors”) arrested estrogentarget tumor cell growth completely at low nanomolar concentrations, andtheir inhibitory effects were entirely reversible by picomolarconcentrations of estrogens. That disclosure revealed a previouslyunknown function for the secretory immune system. In theabove-identified patent applications, a major role for TGFβ in breastgrowth regulation is also identified: it is a cytokine that controlsIgA/IgM immunocytes. Breast cancer growth is best defined as negativeparacrine control by secretory immunoglobulins (immunoglobulininhibitors) and positive direct control by estrogens. In conjunctionwith this work, the longstanding problem of the regulation of estrogendependent cell growth in culture under serum-free defined mediumconditions was solved. These results have great physiological relevance.IgA and IgM are secreted by B immunocytes located in the lamina propriaof estrogen target tissues including breast. They are more than 90% ofthe immunoglobulins secreted into breast milk. The positioning of theimmunocytes in the tissue adjacent to the epithelial cells and thesecretion of the immunoglobulins is hormone regulated.

It was found that the secretory immune system products immunoglobulins Aand M (IgA and IgM) inhibit the growth of estrogen-sensitive (early)breast cancer by suppressing cell replication. This is the first time aconnection was established between the secretory immune system and earlyestrogen receptor positive (ER⁺) breast cancer growth. In addition, whenbreast cancer becomes most malignant (i.e. ER⁻ hormone-insensitivestages), control by immune system IgA and IgM is lost. Evidence pointsto the Poly-Ig receptor or a very similar Poly-Ig-like Fc receptor asthe mediator of the inhibitory effects of IgA and IgM. This receptor maybe the long sought after gene that explains many of the “sporadic”breast cancers. Here, the term “sporadic” refers to breast canceroriginating from unknown genetic origins. The Poly-Ig receptor gene islocated at locus D1S58 on chromosome 1 (23) which has been proven to bea “hot spot” for allelic imbalances in more than 70% of breast cancers(24). A hot spot is a chromosomal loci or gene that is frequentlyaltered in breast cancer specimens. This site has 46% loss ofheterozygosity (LOH) in breast cancer specimens (24) and 30% incidenceof allelic imbalance (AI) (24). This research, and that described in theabove-identified U.S. and PCT patent applications (21,22), support theconclusion that this gene will have notably broader significance in“sporadic” breast cancer etiology than BRCA1 (located on chromosome 17)or BRCA2 (located on chromosome 13).

Mutations in genes that are critical to cell growth or are known to bepredisposing for breast cancer have been described. Such mutations caneither cause activation of oncogenes to promote cell replication orcause inactivation of suppressors to release cells to growth withoutcontrol. In studies related to the present disclosure and described inthe above-identified U.S. and PCT patent applications, the Poly-Ig (Fc)receptor or a similar Poly-Ig-like (Fc) receptor has been identified asa tumor suppressor. Binding the ligands IgA or IgM results in growtharrest in ER⁺ breast cancer cells. When this receptor is absent, cellsreplicate without immune control. The gene identified in this inventionhas not previously been recognized to have growth regulating properties.As this study developed the application of this new receptor genefunction indicated an important concern for both germ line and somaticmutations.

Although the majority of cancers are thought to arise by somaticmutation, the fact that “familial aggregation” of breast cancers existsis strong evidence in favor of germ line analysis. The line of thoughtis to study both types of mutations in the poly-Ig-like receptor. Germline mutations will be sought with blood cells or mucosal cells (i.e.mucosal scrapings) obtained from women. Somatic mutations are revealedvia examination of breast cancer specimens and cells aspirated frombreast ducts or in breast fluid samples.

Key models of germ line breast cancer predisposing genes are readilyavailable from sources known to investigators in this field. Theseinclude TP53, ATM, PTEN, MLH1 and the MSH2 genes. If the full expressionof these predisposing genes is to be realized, other mutations mustcontribute. The Poly-Ig-like receptor is found in every steroid andthyroid hormone responsive cell line examined to date. When the receptoris lost, cells achieve full autonomy. This indicates that the receptorgene may be a key contributor to the development of breast cancer aswell as other cancers arising from mucosal tissues.

EXAMPLE 1 Genetic Analysis of the Poly-Ig (Fc) Receptor or Poly-Ig-like(Fc) Receptor to Determine Breast Cancer Susceptibility in Heterozygotesand Homozygotes

Individuals seeking to determine breast cancer risk for the reasonscited above will be screened for predisposing genetic damage/mutationsin the gene for the Poly-Ig (Fc) receptor or Poly-Ig-like (Fc) receptor.This will be done using lymphocytes and technology suited to rapid butaccurate screening (e.g. pyrosequencing and rapid PCR methodology). Thisanalysis will be preformed similarly to those used to identifyheterozygotes and homozygotes for the ATM mutation and for hereditarynonpolyposis colorectal cancer (HNCC). Studies done with controlvolunteers will be carried out to determine natural innocuous mutationsin the gene and to determine if selected populations have differentmutations and hence may be at greater risk. It is expected that onedamaged gene will confer greater risk than controls because inactivationof the other functional gene will eliminate immune negative growthregulation by IgA and IgM. Suitable techniques that will be used forthis genetic analysis are well known in the art. Such an analysis hasnot been recognized previously as useful in determining risk for breastcancer in populations before development of the disease. Individualsshowing homozygous mutations are considered at highest risk and will becounseled to decide on surgical prophylactic measures or on the use oftamoxifen or other anti-estrogen as preventative measures. Thus, geneticscreening can be used to not only to define potential risk, but toassistant in initiating preventative life saving actions, as discussedin more detail in the following example.

Example 2 Screening for Loss of Heterozygosity (LOH) or AllelicImbalance (AI) in the Poly-Ig (Fc) Receptor or Poly-Ig-like (Fc)Receptor

Using blood cells, mucosal scrapings, breast fluid derived cells andother body and tissue samples and fluids, the presence of LOH and AI inthe Poly-Ig (Fc) receptor or a Poly-Ig-like (Fc) receptor gene will bedetermined by methods commonly applied and well known. Preferably theD1S58 locus of chromosome 1 will be a primary point of focus. Thisanalysis can begin at very young ages (i.e. nine or ten years old) orcan be initiated at age 25 when breast cancer rates are still very low.Analysis of breast fluid cells can be continued with women showing LOHor AI at early ages. This information is used along with methods such asmammography to monitor women at high risk. Early genetic analysis, asdescribed in this example and in Examples 1, 3 and 4, is especiallyvaluable for identifying women at risk so that appropriate steps forrisk reduction or prevention can be taken. If germline mutations arefound or if somatic mutations are found, the issue then becomes “what todo?”. Preventative and therapeutic compositions and methods aredescribed in co-pending U.S. patent application Ser. No. 09/852,547 andin International Patent Application No. PCT/US01/15171 (also identifiedas item 21 in the References, below), or in co-pending U.S. patentapplication Ser. No. ______ (Atty. Dkt. No. 1944-01201) entitled “BreastCancer Eradication Program.” Additional preventative and risk reductionmethods and compositions are described in co-pending U.S. patentapplication Ser. No. ______ (Atty. Dkt. No. 1944-01301) entitled“Anti-estrogen and Immune Modulator Combinations for Treating BreastCancer.” The disclosures of these co-owned patent applications arehereby incorporated herein by reference.

Example 3 Double Screening for Poly-Ig (Fc) Receptor or Poly-Ig-like(Fc) Receptor Mutations and Mutations in other Breast CancerPredisposing Genes

Because the development of cancer most likely depends on more than onemutation (50), and may involve several cell types (50), it is useful toscreen for mutations in genes that will lead to damage in other genes.Both the Lynch Syndrome II genes and the Cowden's disease gene arecandidates for double screening along with the Poly-Ig (Fc) receptor ora Poly-Ig-like (Fc) receptor. For example, changes in the effectivenessof the Lynch Syndrome genes can lead to a gradual accumulation ofmutations. If mutations are also present in the Poly-Ig-like receptor,risk can be expected to be substantially higher than controls.Heterozygotes for the Lynch Syndrome genes have not been previouslyanalyzed to determine if they possess an increased risk of breastcancer. Likewise, heterozygotes for the Cowen's mutation have not beenexamined before to determine breast cancer susceptibility. These resultswill then be compared to results of screens for the Poly-Ig (Fc)receptor or Poly-Ig-like (Fc) receptor to identify those individuals atgreatest risk. Suitable risk reduction or preventative measures can thenbe implemented, as discussed in the preceding Example.

Example 4 Screening for Heterozygous ATM Mutations and Comparison toMutations in the Poly-Ig (Fc) Receptor or Poly-Ig-like Receptor

The ATM mutation, associated with the disorder ataxia telangiectasia, isnot found in breast cancers. Nonetheless, it is clear that evenheterozyogotes are at substantial risk for breast cancer. In light ofthe present disclosure of the importance of breast cancer cell growthcontrol by the secretory immunoglobulins, it is of particularsignificance that the AT disorder is known to be accompanied bychromosome fragility (i.e. lack of repair after ionizing radiation) anda marked deficiency in IgA. Because the ATM mutation is so widelydistributed in the population, it is important to initiate a screeningfor this mutation as a first line defense against breast cancer.Identification of meaningful mutations will then permit decisions bywomen to elect preventative measures. Pending results indicating apotential problem, these same individuals will then be offered anotherscreening for the Poly-Ig (Fc) receptor or a Poly-Ig-like (Fc) receptoras a further indicator of risk status. If significant mutations arefound in one or both receptor genes, prophylactic or preventativemeasures can be initiated, as discussed above in Example 2.

Example 5 Screening of Breast Cancer Specimens for Mutations in GrowthRegulating Genes

Specimens from breast cancer patients will be screened for alterationsin the Poly-Ig (Fc) receptor or a Poly-Ig-like receptor to assist intherapy decisions and to identify individuals at greatest risk andrequiring more intense intervention. This approach identifies somaticmutations. Conventional screening techniques will also be used toidentify heterozygous genes including the Lynch Syndrome gene and theCowden's disease gene as well as alterations in TP53. The molecularfingerprinting of tumors is expected to increase the effectiveness oftreatment programs by allowing each to be adapted to the individualpatient. Future use of molecular methods is expected to provide agenetic profile of a patient's primary tumor as well as to provideinformation relevant to family members concerning their potential risks.

Example 6 Evaluation of Breast Fluid Derived Cells for Molecular Changesin Genes

Cells will be obtained from breast fluids by any of a number of wellknown methods, or, alternatively, by newer methods that are known in theart and have been described in the literature for direct aspiration ofthe breast milk ducts. Commercial milk pumps are available for thisapplication. Premalignant changes in the Poly-Ig (Fc) receptor or aPoly-Ig-like (Fc) receptor gene will be evaluated as will be changes inthe other breast cancer predisposing genes discussed above. Thistechnology is expected to identify somatic mutations at the site ofbreast cancer development. Identification of sets of changes consistentwith long-term development of the disease will permit immediateintervention to eradicate the altered cells or arrest the mutationprocess.

After parturition, cells will be harvested from expressed breast milkand evaluated for somatic mutations. This analysis can be readilycarried out during routine postpartum doctor's office visits. The cellswill be collected directly on to filters (supplied) and DNA screeningconducted, as previously described. This method permits directassessment of the genetic status of a subset of reproductive age womenwithout disruption of daily routines. Fluid aspiration can also be doneduring routine mammography examinations. Nipple pumps, which areavailable from well known commercial suppliers, can be used to withdrawonly the few milliliters of fluid required.

Once a person or a group of persons have been identified as being atrisk for breast cancer using one or more of the foregoing procedures, aprogram of prevention or risk reduction can be implemented, as describedin, for example, co-pending U.S. patent application Ser. No. ______(Atty. Dkt. No. 1944-01201) entitled “Breast Cancer EradicationProgram,” and U.S. patent application Ser. No. ______ (Atty. Dkt. No.1944-01301) entitled “Anti-estrogen and Immune Modulator Combinationsfor Treating Breast Cancer.” The disclosures of these co-owned patentapplications are hereby incorporated herein by reference. Such programcan include (a) enhancing the amount of an immunoglobulin inhibitor(“immune modulator”) of cancer cell growth that contacts the breastductal tissue of said individuals; and/or administering animmunoglobulin inhibitor mimicking compound; administering ananti-estrogenic compound; (b) administering an aromatase inhibitor; (c)enhancing the number of B immunocytes producing IgA or IgM in breasttissue; and/or (d) immunizing individuals at risk of developing breastcancer against microorganisms known to or suspected of causing breastcancer. Some immunoglobulin inhibitor mimicking compounds that may beused include: Tamoxifen and MER-25 and chemically substituted ormodified derivatives thereof. To reduce possible side effects of MER-25,or its derivative compound, in some cases it may also be desirable toco-administer progesterone or another hormone. Some immune enhancersthat may be used include: Levimisole, Imiquimod, Picibanil, and DHEA.Some useful anti-estrogens include: Tamoxifen, Toremifene, ICI 16384,ICI 182780, EM-800, RU 58688 and EM-139.

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While the preferred embodiments of the invention have been shown anddescribed, modifications thereof can be made by one skilled in the artwithout departing from the spirit and teachings of the invention. Theembodiments described herein are exemplary only, and are not intended tobe limiting. Many variations and modifications of the inventiondisclosed herein are possible and are within the scope of the invention.The disclosures of all patents, patent applications and publicationscited herein are hereby incorporated herein by reference. The discussionof certain references in the Description of Related Art, above, is notan admission that they are prior art to the present invention,especially any references that may have a publication date after thepriority date of this application.

1-27. (canceled)
 28. A method of treating an individual identified as heterozygous or homozygous for a mutation in a gene coding for a Poly-Ig (Fc) receptor or a Poly-Ig-like (Fc) receptor capable of mediating estrogen reversible inhibition of breast cancer cell growth by an immunoglobulin inhibitor, comprising enhancing the amount of immunoglobulin inhibitor contacting a mucosal epithelial tissue of said individual.
 29. The method of claim 28, wherein said individual is identified as homozygous for said mutation, the method comprising prophylactic administration to said individual of an anti-estrogen compound.
 30. The method of claim 29, wherein said anti-estrogen compound is chosen from the group consisting of Tamoxifen, Toremifene, ICI 16384, ICI 182780, EM-800, RU 58688 and EM-139.
 31. The method of claim 28, wherein said individual is identified as homozygous for said mutation, the method comprising prophylactic surgical removal of non-cancerous breast tissue.
 32. The method of claim 28, comprising employing a breast cancer risk reduction or prevention program comprising enhancing the amount of immunoglobulin inhibitor of cancer cell growth that contacts the breast ductal tissue of said individuals, and/or administering an immunoglobulin inhibitor mimicking compound; and at least one of the following treatments: (a) administering an anti-estrogenic compound; (b) administering an aromatase inhibitor; (c) enhancing the number of B immunocytes producing IgA or IgM in breast tissue; (d) immunizing individuals at risk of developing breast cancer against microorganisms known to or suspected of causing breast cancer.
 33. The method of claim 32, wherein said immunoglobulin inhibitor mimicking compound is chosen from the group consisting of Tamoxifen, MER-25 and chemically substituted or modified derivatives thereof.
 34. The method of claim 33, comprising co-administering a hormone capable of reducing side effects of said MER-25 or derivative thereof
 35. The method of claim 32, wherein enhancing the number of B immunocytes producing IgA or IgM in breast tissue comprises administering an immune enhancing agent.
 36. The method of claim 35, wherein said immune enhancing agent is chosen from the group consisting of Levimisole, Imiquimod, Picibanil and DHEA. 